A DNA Damage-Induced, SOS-Independent Checkpoint Regulates Cell Division in Caulobacter crescentus

Cells must coordinate DNA replication with cell division, especially during episodes of DNA damage. The paradigm for cell division control following DNA damage in bacteria involves the SOS response where cleavage of the transcriptional repressor LexA induces a division inhibitor. However, in Caulobacter crescentus, cells lacking the primary SOS-regulated inhibitor, sidA, can often still delay division post-damage. Here we identify didA, a second cell division inhibitor that is induced by DNA damage, but in an SOS-independent manner. Together, DidA and SidA inhibit division, such that cells lacking both inhibitors divide prematurely following DNA damage, with lethal consequences. We show that DidA does not disrupt assembly of the division machinery and instead binds the essential division protein FtsN to block cytokinesis. Intriguingly, mutations in FtsW and FtsI, which drive the synthesis of septal cell wall material, can suppress the activity of both SidA and DidA, likely by causing the FtsW/I/N complex to hyperactively initiate cell division. Finally, we identify a transcription factor, DriD, that drives the SOS-independent transcription of didA following DNA damage.National Institutes of Health (U.S.) (Grant R01GM082899)National Science Foundation (U.S.). Graduate Research Fellowship Progra

A DNA damage checkpoint in Caulobacter crescentus inhibits cell division through a direct interaction with FtsW

Following DNA damage, cells typically delay cell cycle progression and inhibit cell division until their chromosomes have been repaired. The bacterial checkpoint systems responsible for these DNA damage responses are incompletely understood. Here, we show that Caulobacter crescentus responds to DNA damage by coordinately inducing an SOS regulon and inhibiting the master regulator CtrA. Included in the SOS regulon is sidA (SOS-induced inhibitor of cell division A), a membrane protein of only 29 amino acids that helps to delay cell division following DNA damage, but is dispensable in undamaged cells. SidA is sufficient, when overproduced, to block cell division. However, unlike many other regulators of bacterial cell division, SidA does not directly disrupt the assembly or stability of the cytokinetic ring protein FtsZ, nor does it affect the recruitment of other components of the cell division machinery. Instead, we provide evidence that SidA inhibits division by binding directly to FtsW to prevent the final constriction of the cytokinetic ring.Howard Hughes Medical Institute (Early Career Scientist)National Institutes of Health (U.S.) (NIH grant (5R01GM082899))National Institutes of Health (U.S.) (Predoctoral Fellowship

Prophage-like gene transfer agents promote Caulobacter crescentus survival and DNA repair during stationary phase

Gene transfer agents (GTAs) are prophage-like entities found in many bacterial genomes that cannot propagate themselves and instead package approximately 5 to 15 kbp fragments of the host genome that can then be transferred to related recipient cells. Although suggested to facilitate horizontal gene transfer (HGT) in the wild, no clear physiological role for GTAs has been elucidated. Here, we demonstrate that the α-proteobacterium Caulobacter crescentus produces bona fide GTAs. The production of Caulobacter GTAs is tightly regulated by a newly identified transcription factor, RogA, that represses gafYZ, the direct activators of GTA synthesis. Cells lacking rogA or expressing gafYZ produce GTAs harboring approximately 8.3 kbp fragment of the genome that can, after cell lysis, be transferred into recipient cells. Notably, we find that GTAs promote the survival of Caulobacter in stationary phase and following DNA damage by providing recipient cells a template for homologous recombination-based repair. This function may be broadly conserved in other GTA-producing organisms and explain the prevalence of this unusual HGT mechanism.</jats:p

Two independent pathways regulate cell division in <i>Caulobacter</i> following DNA damage.

<p>(A–B) Two cell division inhibitors are induced following DNA damage in <i>Caulobacter</i>. <i>sidA</i> is induced by cleavage of the SOS repressor LexA while <i>didA</i> is induced by DriD. SidA and DidA are small transmembrane proteins that can block cell division by preventing the divisome subcomplex FtsW/I/N from assuming an active state, designated FtsW/I/N*. FtsW/I/N* could promote division by enhancing peptidoglycan synthesis and remodeling, by triggering FtsZ constriction, or by coordinating these activities.</p

Cells lacking <i>sidA</i> and <i>didA</i> cannot properly regulate cell division following DNA damage.

<p>(A) Wild-type, <i>ΔsidA</i>, <i>ΔdidA</i>, and <i>ΔsidAΔdidA</i> cells were grown to mid-exponential phase and plated in 10-fold dilutions on rich media with or without 0.35 µg/ml MMC. (B) Wild-type and <i>ΔsidAΔdidA</i> cells carrying an empty plasmid, and <i>ΔsidAΔdidA</i> cells carrying a plasmid with either <i>sidA</i> or <i>didA</i> driven by its native promoter were plated as in (A). (C–E) Synchronous populations of swarmer cells from the strains in (A) were placed on agarose pads containing rich media and MMC and imaged for 8 hours by time-lapse microscopy. (C) The time to first mid-cell division and (D) the percentage of cells that stopped growing following division relative to the wild type are shown (for criteria on calling divisions and growth cessation, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001977#pbio.1001977.s015" target="_blank">Text S1</a>). The data in (C) are representative of biological duplicates. The data in (D) are averaged from biological duplicates. Asterisks represent a statistically significant (<i>p</i><0.01) difference relative to the wild type. Error bars represent standard error of the mean (SEM). (E) Representative fields of wild-type and <i>ΔsidAΔdidA</i> swarmer cells grown on pads containing MMC at the time points indicated in hours. Black arrows indicate cells that divided. Gray arrows indicate cells arrested for growth following division. Bar, 2 µm.</p

Mutations that suppress <i>sidA</i> and <i>didA</i> overexpression likely hyperactivate cell division.

<p>(A) The strains indicated were grown to mid-exponential phase in rich media and imaged by phase microscopy. Bar, 2 µm. (B) Each strain indicated was grown to mid-exponential phase and average cell length, relative to wild-type, was calculated (all <i>n</i>>440). Error bars represent standard error of the mean (SEM), and asterisks indicate <i>p</i><0.01 (*) or <i>p</i><0.0001 (**). The strain denoted <i>ftsW**I*</i> combines the mutations <i>ftsW(F145L, A246T)</i> and <i>ftsI(I45V)</i>. Separate graphs are shown for cell length measurements made on different days. For raw data, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001977#pbio.1001977.s014" target="_blank">Data S3</a>. (C) Wild-type, <i>ΔsidAΔdidA</i>, <i>ftsW(A246T)</i>, and <i>ftsW**I*</i> cells were grown to mid-exponential phase and plated in 10-fold dilutions on rich media containing no additives, 0.35 µg/ml MMC or 6 µg/ml cephalexin. (D) The strains from (C) were grown to mid-exponential phase in rich media and treated with MMC or cephalexin at the concentrations in (C) for 6 hours. PI at 5 µM was added 1.5 hours before imaging. Cells were imaged by phase and fluorescence microscopy; cell lengths and percentage of PI+ cells are shown by bar graphs. For raw data, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001977#pbio.1001977.s014" target="_blank">Data S3</a>.</p

DidA is a small, inner membrane protein that interacts with FtsN.

<p>(A) The subcellular localization of DidA was examined in a strain expressing <i>M2-yfp-didA</i> from the xylose-inducible promoter P<i>_xyl</i> on a low-copy plasmid. Cells were grown to mid-exponential phase in rich media with glucose and then shifted to xylose. At the times indicated, cells were imaged by phase and epifluorescent microscopy. In the fluorescent micrographs, cell boundaries were added after imaging. (B) Subcellular fractionation of cells overexpressing <i>3×M2-didA</i> from the P<i>_van</i> promoter on a medium-copy plasmid for 1.5 hours and expressing the transmembrane protein <i>cckA-gfp</i> from P<i>_cckA</i> on the chromosome. Samples were fractionated into soluble (S) and membrane (M) fractions and analyzed by Western blot. The membrane was cut into three pieces, indicated by dashed lines, and probed with antibodies specific for the GFP, CtrA, or M2 epitope. (C) Bacterial two-hybrid analysis of interactions between T25-DidA and cell division proteins fused to T18, as indicated. The FtsIΔC construct lacking the C-terminal catalytic domain previously showed interactions with FtsW and FtsN as expected, unlike the full-length version of FtsI <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001977#pbio.1001977-Modell1" target="_blank">[13]</a>. The interacting pair T18-M2-SidA and T25-FtsN was included for comparison. <i>E. coli</i> strains harboring each pair of fusions were plated on LB, and colonies were restruck on MacConkey plates containing maltose. Red streaks indicate positive interactions. −/− indicates empty vectors negative control, +/+ indicates the zip/zip fusions used as a positive control. (D) Subcellular localization of FtsZ, FtsW, FtsI, and FtsN were examined in strains expressing <i>ftsZ-yfp</i> from the chromosomal P<i>_van</i> promoter, or <i>venus-ftsW</i>, <i>gfp-ftsI</i> or <i>gfp-ftsN</i> from its native chromosomal locus. Each strain was transformed with a medium-copy plasmid expressing <i>didA</i> from the P<i>_van</i> promoter. Strains were grown to mid-exponential phase and samples imaged by phase and epifluorescent microscopy after addition of vanillate for 4.5 hours. In the fluorescent images, cell outlines were drawn based on the phase micrographs. Bar, 2 µm. (E) Strains from (D) were grown to mid-exponential phase and 10-fold serial dilutions were plated on rich media supplemented with vanillate to induce <i>didA</i> expression.</p

DriD directly activates <i>didA</i>.

<p>(A) Wild-type cells harboring low-copy plasmids expressing <i>egfp</i> from the <i>sidA</i> or <i>didA</i> promoters were treated with MMC (0.5 and 3 µg/ml), hydroxyurea (HU; 0.5 and 3 mg/ml) or zeocin (2.5 and 15 µg/ml) and then analyzed by Western blot using an α-GFP antibody. (B) Diagram of <i>driD</i> indicating the predicted helix-turn-helix (HTH) and WYL domains. Arrows indicate transposon insertion sites in the genetic screen that identified <i>driD</i>. (C) Wild-type, <i>ΔdriD</i>, and <i>ΔrecA</i> cells were transformed with the P<i>_sidA</i> and P<i>_didA</i> reporter plasmids from (A) and treated with 3 µg/ml MMC or 15 µg/ml zeocin for 1 hour. Samples were analyzed by Western blot using an α-GFP antibody. (D) 10-fold serial dilutions of the strains indicated were grown on plates containing 0.35 µg/ml MMC. (E) <i>ΔdriD</i> cells carrying a low-copy plasmid producing a control construct (P<i>_xyl-ftsW-egfp</i>), untagged DidA, or DidA fused at either its N- or C-terminal end to a 3×M2 tag and expressed from the <i>didA</i> promoter were treated with 15 µg/ml zeocin for 45 minutes. Samples were analyzed by Western blot using an α-FLAG/M2 antibody. (F) <i>ΔdriD</i> cells carrying a low-copy plasmid expressing either <i>driD</i> or <i>driD-3×M2</i> from the <i>driD</i> promoter were treated with 15 µg/ml zeocin for 45 minutes. DriD was immunoprecipitated with an α-FLAG/M2 antibody and promoter occupancy was analyzed by quantitative PCR using primers specific for P<i>_didA</i>. Fold-enrichment values were normalized relative to the enrichment of a region within the coding sequence of <i>ruvA</i>. For raw data, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001977#pbio.1001977.s014" target="_blank">Data S3</a>.</p

<i>didA</i> is induced by DNA damage and is not SOS regulated.

<p>(A) Wild-type and Δ<i>recA</i> cells were grown in rich medium to mid-exponential phase and treated with 1 µg/ml MMC for 30 minutes. Expression values, the average of two biological replicates, are shown for the 50 most upregulated genes in wild-type cells with fold-change ratios calculated in comparison to mock treated cells. The dashed line corresponds to fold-change values that are identical in wild-type and <i>ΔrecA</i> cells. For complete data, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001977#pbio.1001977.s002" target="_blank">Figure S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001977#pbio.1001977.s012" target="_blank">Data S1A</a>. (B) CC3114 and CCNA03212 (<i>didA</i>) are shown schematically in their genomic context. Nucleotide positions relative to the annotated CC3114 start site are shown below. The gray shaded region represents a predicted transmembrane domain. (C) Western blot of cells producing DidA fused to a C-terminal 3×M2 epitope from the chromosomal <i>didA</i> locus. Cells were grown to mid-exponential phase and treated with 1 µg/ml MMC for the times indicated. (D) Western blot of wild-type, Δ<i>recA</i> and <i>lexA(K203A)</i> cells expressing <i>didA-3×M2</i> from its native locus treated with 1 µg/ml MMC for 1 hour. Membranes (C–D) were blotted with the α-FLAG/M2 antibody.</p